The invention relates to the electrophoresis process wherein biological cells, colloidal particles, or macromolecules with a net electrical charge migrate and separate in a solution under the influence of an electrical field and, more particularly, to improved apparatus for carrying out such a process.
Heretofore, conventional continuous electrophoretic separation devices have utilized a flat rectangular separation chamber to contain a flowing curtain of buffer in which a specimen is injected and where the separation process is conducted. An electrical field across the width of the chamber causes the injected sample cells to be separated into fraction bands through differential migration of the sample fractions. An array of collection receptacles located along the lower portion of the chamber collects the fractionated bands and hence provides for a continuous separation and collection of injected sample.
However, when fluid flows in a rectangular chamber, a parabolic velocity profile (Poiseuille flow) is established across the thickness of the chamber due to the no-slip boundary condition at the chamber walls. The Poiseuille flow of the buffer curtain causes sample particles distributed throughout the curtain thickness to have varying residence times in the chamber. This causes the deflection of a particle near the curtain midplane to be less than that for a similar particle near the chamber wall, providing distortion (crescent formation) of an initially straight injected sample band.
Adding to flow distortion is an effect known as electroosmosis which, as opposed to Poiseuille flow, causes a slip condition to occur at the chamber wall which produces an effect opposite to that of the Poiseuille distortion. Electroosmosis is a lateral flow across the width of the chamber which exists when charged walls are present.
As a result of laminar flow, a particle traveling through the separation chamber at or near the center plane will be deflected less than an electrophoretical similar particle moving through at some distance from the center plane. Therefore, an initially regular pattern of injected sample will be distorted into a convex shape when viewed in the direction of sample migration. On the other hand, electroosmotic flow increases the deflection of particles at on near the center plane so that concave patterns are formd when viewed in the direction of sample migration. These phenomena combine to produce crescent-shaped distortions which produce a variation in resolution across the chamber collection width. The curvature of the crescent-shaped distortions is determined by the flow that predominates--laminar flow or electroosmosis. Therefore, unless exact compensation exists, a bending of the injected sample band will result--an artifact which we will call flow distortion. The condition for exact compensation for these flow effects is the equality of sample and wall zeta potential. Maximum resolution occurs for an equality of charge (zeta potential) between the chamber walls and specific sample fraction. Since the wall usually has only one zeta potential, only one fraction can be in focus. A more thorough discussion of this phenomena may be found in Strickler, A. and Sacks, T. Preparative Biochemistry, 3, p. 269-277 (1973).
Due to the difficulty encountered in nullifying the above mentioned distortions, the sample stream is usually injected only in the center plane region of the chamber thickness. This practice is not only inefficient in the use of the chamber volume, but it is also a very unreliable method to control distortion because there are no positive means assuring maintenance of the sample near the chamber center plane. Therefore, during actual operation of a conventional continuous flow device, the sample deviates from the center plane region of buffer to marginal zones, establishing a limit on resolution.
An inherent disadvantage of any electrophoresis process is its inability to self-sharpen or self-stabilize the fraction bands during separation. The self-sharpening ability is called "focusing" and an example of which is isoelectric focusing. Not only does the focusing capacity lead to reliable long term processing durations, but it also increases throughput by allowing sample to be injected throughout the entire chamber volume.
Prior electrophoresis devices have been proposed for operation in space such as disclosed in Bier et al., "Preparative Electrophoresis in Zero Gravity," Journal of Colloid and Interface Science, 55, No. 1 (Apr. 1976) and Strickler, "Deflected-Laminar Electrophoresis," American Institute of Aeronautics and Astronautics Paper No. 77-233 (1977). However, these devices were intended only to improve throughout and not resolution. Most methods proposed to date for solving the flow distortion problem have merely sought to compensate for the disturbance rather than remove it. Operating in such a manner is rather like a balancing act and is not conducive to reliable operation. In addition, only one component of the sample will be in "focus" at any one time.
The thick chamber for use in zero gravity is a method which does not involve compensation. The increase in resolution would be achieved by keeping the sample away from the chamber walls via the increased chamber thickness and, hence, reduce sample band distortion. However, the resolution decreases with increasing chamber thickness for constant power dissipation in the chamber because the increase in thickness will also dictate a lower applied voltage for constant mid-plane temperature. Thus, merely increasing the chamber thickness without considering the impact on other operational parameters does not increase the resolution of separation.
Another method to eliminate flow disturbances was advanced in Kolin, A., Ellerbroek, B. L., "Theory of Simultaneous Multiple Streak Collimation in Continuous-Flow Electrophoresis by Superposition of Electro-Osmosis and Thermal Convection," Separation and Purification Methods 8, 1-19 (1979). This method, which might be thought of as the ultimate in compensation, uses a cross flow to neutralize electroosmosis and relies on thermal convection to blunt the parabolic flow-through profile sufficiently so that the center-plane region of the chamber will be distortion free. The power levels necessary to cause the required deformation of the parabolic flow-through profile are many times the power level limit that will disrupt electrophoresis in conventional separation chambers. Also, the exact and uniform counter flow along the length of the chamber necessary to counter electroosmosis would be very difficult to achieve. While this scheme is possible theoretically, it is practically impossible to implement successfully.
Probably the most practical idea yet employed to compensate for chamber flow distortions was developed in Strickler, A., Sacks, T., "Focussing in Continuous Flow Electrophoresis System by Electrical Control of Effective Cell Wall Zeta Potential." Annals of New York Academy of Science 209 (1973). The concept consisted of coating longitudinal sections of the inner chamber wall with materials having different zeta potentials and sectioning the respective electrodes so that the electrical field could be independently applied to each section. By controlling the electrical field strength in each section, flow distortions (crescent formations) created in a previous section could be compensated in a subsequent section simply by turning a control knob to change the applied voltage. The "focusing" process, however, had to be controlled by visual observation through a cross-section illuminator which revealed the crescent-shaped band cross sections. Although this concept is theoretically sound and workable, it has not found great acceptance in the field. It is impractical that the system requires a constant operator interface to maintain precise "focusing." Small changes in zeta potential matching can cause large changes in resolution.
It, therefore, appears that new methods and design concepts must be brought to overcome the problem of sample stream distortion, leading to a new device which would offer unique capabilities for operation, particularly in reduced gravity, and offer a significant improvement in resolution over similar ground-based machines. As proposed herein, these objectives are met by a concept and apparatus which utilize moving separation chamber walls. The moving walls entrain the fluid to flow as a rigid body, hence eliminating Poiseuille flow. All of the sample throughout the chamber thickness is thus exposed to the imposed electric field for the same period of time, while electroosmosis has been eliminated through the use of film-forming latexes. The zeta potential of the latex has been altered by prior coating of the particle surface with Methylcellulose which has been found to yield a zeta potential near zero. Since both sources for the disturbances have been eliminated, no compensation is required. The system operates like a static device while providing throughput like a conventional continuous-flow system. In addition, no limitation is placed on the usable fraction of the chamber thickness.
Accordingly, an important object of the present invention is to advance the state-of-the art in continuous electrophoresis devices and specifically to increase the resolution and throughput of such devices by the elimination of distortion problems associated therewith.
Another important object of the present invention is to provide a continuous electrophoresis device in which the buffer and specimen flow is generally static with respect to walls of the separation chamber.
Another important object of the present invention is to provide a static continuous electrophoresis device in which laminar flow distortion of the sample bands is virtually eliminated.
Yet another important object of the present invention is to provide a static continuous electrophoresis device in which electroosmosis is eliminated.
Yet another important object of the present invention is to provide a device which will optimize performance in such a manner as to make processing of biological cells feasible in space.